Int. J. Mol. Sci. 2009, 10, 2440-2475; doi:10.

3390/ijms10062440

International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
Review
An Updated Review of Tyrosinase Inhibitors
Te-Sheng Chang
Department of Biological Science and Technology, National University of Tainan, 33 sec. 2 Shu-Lin
St., Tainan, Taiwan; E-Mail: mozyme2001@yahoo.com.tw; Tel. +886 6 2606283;
Fax: +886 6 2909502
Received: 21 April 2009; in revised form: 8 May 2009 / Accepted: 21 May 2009 /
Published: 26 May 2009

Abstract: Tyrosinase is a multifunctional, glycosylated, and copper-containing oxidase,
which catalyzes the first two steps in mammalian melanogenesis and is responsible for
enzymatic browning reactions in damaged fruits during post-harvest handling and
processing. Neither hyperpigmentation in human skin nor enzymatic browning in fruits are
desirable. These phenomena have encouraged researchers to seek new potent tyrosinase
inhibitors for use in foods and cosmetics. This article surveys tyrosinase inhibitors newly
discovered from natural and synthetic sources. The inhibitory strength is compared with
that of a standard inhibitor, kojic acid, and their inhibitory mechanisms are discussed.
Keywords: browning; inhibitors; melanogenesis; tyrosinase

1. Introduction
For the past few decades, tyrosinase inhibitors have been a great concern solely due to the key role
of tyrosinase in both mammalian melanogenesis and fruit or fungi enzymatic browning. Melanogenesis
has been defined as the entire process leading to the formation of dark macromolecular pigments, i.e.,
melanin. Melanin is formed by a combination of enzymatically catalyzed and chemical reactions. The
biosynthetic pathway for melanin formation in various life forms has firstly been elucidated by Raper
[1], Mason [2] and recently been modified by Cooksey et al. [3] and Schallreuter et al. [4] (Figure 1).
Melanogenesis is initiated with the first step of tyrosine oxidation to dopaquinone catalyzed by
tyrosinase. This first step is the rate-limiting step in melanin synthesis because the remainder of the
reaction sequence can proceed spontaneously at a physiological pH value [5]. The subsequent
dopaquinone is converted to dopa and dopachrome through auto-oxidation. Dopa is also the substrate
OPEN ACCESS
Int. J. Mol. Sci. 2009, 10


2441
of tyrosinase and oxidized to dopaquinone again by the enzyme. Finally, eumelanin are formed
through a series of oxidation reactions from dihydroxyindole (DHI) and dihydroxyindole-2-carboxylic
acid (DHICA), which are the reaction products from dopachrome. In the presence of cysteine or
glutathione, dopaquinone is converted to cysteinyldopa or glutathionyldopa. Subsequently,
pheomelanin is formed. In addition to eumelanin and pheomelanin, other melanin relying on
phenolic monomers different from tyrosine is termed allomelanin. The browning phenomenon in fruit
and fungi is also usually related to oxidative polymerization, conceptually similar to melanogenesis.
The main difference resides in the fact that allomelanin substantially does not contain dopaquinone-
derived motifs as the main monomers in its structure and, on the contrary, is based on other quinoid
building blocks. Melanin plays an important role in protecting human skin from the harmful effects of
UV radiation from the sun. Melanin also determines our phenotypic appearance.
Figure 1. Biosynthetic pathway of melanin [1-4]. TYR, tyrosinase; TRP; tyrosinase related
protein; dopa, 3,4-dihydroxyphenylalanine; DHICA, 5,6-dihydroxyindole-2-carboxylic
acid; DHI, 5,6-dihydroxyindole; ICAQ, indole-2-carboxylic acid-5,6-quinone; IQ, indole-
5,6-quinone; HBTA, 5-hydroxy-1,4-benzothiazinylalanine.
NH
2
COOH
O H
TYR
NH
2
COOH
O H
O H
Tyrosine
Dopa
T
Y
R
NH
2
COOH
O
O
Dopaquinone
N
H
O H
O H
COOH
Leukodopachrome
O
O
N
H
COOH
ICAQ
NH+ O
COOH
O
Dopachrome
TRP-2
TRP-1
N
H
O H
O H
COOH
-CO
2
N
H
O H
O H
DHI
TYR
O
O
N
H
DHICA
IQ
Eumelanin
Glutathione or Cysteine
NH
2
COOH
O H
O H
S
N H
2
COOH
N
S
NH
2
COOH O H
Pheomelanin
Cysteinyldopa
HBTA
Mixed-melanin
Eumelanogenesis Pheomelanogenesis
NH
2
COOH
O H
TYR
NH
2
COOH
O H
O H
Tyrosine
Dopa
T
Y
R
NH
2
COOH
O
O
Dopaquinone
N
H
O H
O H
COOH
Leukodopachrome
O
O
N
H
COOH
ICAQ
NH+ O
COOH
O
Dopachrome
TRP-2
TRP-1
N
H
O H
O H
COOH
-CO
2
N
H
O H
O H
DHI
TYR
O
O
N
H
DHICA
IQ
Eumelanin
Glutathione or Cysteine
NH
2
COOH
O H
O H
S
N H
2
COOH
N
S
NH
2
COOH O H
Pheomelanin
Cysteinyldopa
HBTA
Mixed-melanin
Eumelanogenesis Pheomelanogenesis


Although melanin has mainly a photoprotective function in human skin, the accumulation of an
abnormal amount of melanin in different specific parts of the skin resulting in more pigmented patches
might become an esthetic problem. In addition, enzymatic browning in fruit and fungi is undesirable in,
for example, fresh fruits, beverages, vegetables, and mushrooms [6]. Browning after harvest is a
common phenomenon in crops such as mushrooms, which decreases the commercial value of the
products. Hyperpigmentation in human skin and enzymatic browning in fruits are not desirable. These
phenomena have encouraged researchers to seek new potent tyrosinase inhibitors for use in
Int. J. Mol. Sci. 2009, 10


2442
antibrowning of foods and skin whitening. Some tyrosinase inhibitors have been discovered and
reviewed before [7-9]; this article surveys tyrosinase inhibitors newly discovered from natural
and synthetic sources.
On the other hand, knowledge of melanocyte biology and the processes underlying melanin
synthesis has made remarkable progress over the last few years, opening new paths in the
pharmacologic approach to the treatment of skin hyperpigmentation. In addition to inhibition
of tyrosinase catalytic activity, other approaches to treat hyperpigmentation include inhibition of
tyrosinase mRNA transcription, aberration of tyrosinase glycosylation and maturation, acceleration
of tyrosinase degradation, interference with melanosome maturation and transfer, inhibition of
inflammation-induced melanogenic response, and acceleration of skin turnover. Accordingly, a huge
number of depigmenting agents or whitening agents developed by those alternative approaches have
been successfully identified and deeply reviewed in many articles [10-16]. Hence, these
multidirectional approaches to treat hyperpigmentation are not discussed in this review.
2. Biochemical Characteristics and Reaction Mechanism of Tyrosinase
A number of research papers and reviews have already been published on the structural and kinetic
aspects of the enzyme tyrosinase [17-22]; therefore, under this section I will briefly discuss
tyrosinases biochemical characteristics and reaction mechanism.
Tyrosinases (EC 1.14.18.1) catalyze the oxidations of both monophenols (cresolase or
monophenolase activity) and o-diphenols (catecholase or diphenolase activity) into reactive
o-quinones. The term tyrosinase refers to its typical substrate, tyrosine. Both tyrosinase activities
appear to have broad substrate specificities, although the enzyme has a higher affinity for the
L-isomers of the substrates than for the corresponding D-isomers. The first biochemical investigations
were carried out in 1895 on the mushroom Russula nigricans, whose cut flesh turns red and then black
on exposure to air. Since this study, the enzyme has been found widely distributed throughout the
phylogenetic scale from bacteria to mammals. The best-characterized tyrosinases are derived from
Streptomyces glausescens, the fungi Neurospora crassa and Agaricus bisporus. In fungi and
vertebrates, tyrosinase catalyzes the initial step in the formation of the pigment melanin from tyrosine.
In plants, the physiological substrates are a variety of phenolics. Tyrosinase oxidizes them in the
browning pathway observed when tissues are injured. The enzyme extracted from the champignon
mushroom A. bisporus is highly homologous with the mammalian ones, and this renders it well suited
as a model for studies on melanogenesis. In fact, almost all studies on tyrosinase inhibition conducted
so far have used mushroom tyrosinase because the enzyme is commercially available.
The notable feature observed in tyrosinases from different sources is that the central copper-binding
domain is conserved, which contains strictly conserved amino acid residues, including three histidines
[17,23-24]. One tyrosinase molecule can contain two copper atoms, and each atom of the binuclear
copper cluster is ligated to three histidines. In the formation of melanin pigments, three types of
tyrosinase (oxy-, met-, and deoxytyrosinase, Figure 2) with different binuclear copper structures of the
active site are involved. The oxygenated form (oxytyrosinase, E
oxy
) consists of two tetragonal copper
(II) atoms, each coordinated by two strong equatorial and one weaker axial N
His
ligand. The exogenous
oxygen molecule is bound as peroxide and bridges the two copper centers. Mettyrosinase (E
met
),
Int. J. Mol. Sci. 2009, 10


2443
similar to the oxy form, contains two tetragonal copper (II) ions coupled through an endogenous
bridge, although hydroxide exogenous ligands other than peroxide are bound to the copper site.
Deoxytyrosinase (E
deoxy
) contains two copper (I) ions with a co-ordination arrangement similar to that
of the met form, but without the hydroxide bridge. The resting form of tyrosinase, i.e., the enzyme as
obtained after purification, is found to be a mixture of 85% met and 15% oxy forms.
Figure 2. Catalytic cycles of the hydroxylation of monophenol and oxidation of o-diphenol
to o-quinone by tyrosinase [23-24]. E
oxy
, E
met
, and E
deoxy
are the three types of tyrosinase,
respectively. E
oxy
D, E
oxy
M, and E
met
M are E
oxy
-Diphenol, E
oxy
-Monophenol, and E
met
-
Monophenol complexes, respectively.
R
OH
O H
R
OH
E
oxy
E
oxy
D
E
met
E
met
D
E
deoxy
E
oxy
M
O
2
R
OH
O H
E
met
M
R
OH
Monophenolase
Catalytic Cycle
Diphenolase
Catalytic Cycle
O
R
O
O
R
O
R
OH
O H
R
OH
E
oxy
E
oxy
D
E
met
E
met
D
E
deoxy
E
oxy
M
O
2
R
OH
O H
E
met
M
R
OH
Monophenolase
Catalytic Cycle
Diphenolase
Catalytic Cycle
O
R
O
O
R
O


The above considerations have led to the molecular mechanism for the monophenolase and
diphenolase activities of tyrosinase (Figure 2). In the monophenolase cycle, the monophenol can react
only with the oxy form and be oxidized to the o-quinone, resulting in a deoxy form ready for further
dioxygen binding. Oxytyrosinase is, then, regenerated after the binding of molecular oxygen to
deoxytyrosinase. If only o-diphenol is present (the diphenolase cycle), both the oxy and met forms
react with o-diphenol, oxidizing it to the o-quinone. o-Diphenol binds to the oxy form and is oxidized
to o-quinone, yielding the met form of the enzyme. The latter form transforms another o-diphenol
molecule into o-quinone and is reduced to the bicuprous deoxy form. In most situations, a diphenol is
necessary as the reducing agent to obtain the deoxy form, the only one capable of reacting with
molecular oxygen and continuing in the catalytic action. For this reason, the monophenolase activity
presents a characteristic lag time that exists until a sufficient amount of catechol (needed to reduce the
met form to the deoxy one) is produced by the small amount of the oxy form generally present in the
resting enzyme preparations. The length of the lag time depends on several factors: the enzyme source;
the concentration of monophenol (the lag period being longer when monophenol concentration is
increased); the enzyme concentration (with the lag period diminishing, but never totally disappearing,
Int. J. Mol. Sci. 2009, 10


2444
when the enzyme concentration is increased); and finally, the presence of catalytic amounts of
o-diphenol or transition metal ions, which completely abolish the lag period.
3. Tyrosinase Inhibitors
A number of tyrosinase inhibitors from both natural and synthetic sources have been identified.
However, the definition of tyrosinase inhibitor is sometimes misleading: many authors use that
terminology in reference to melanogenesis inhibitors, whose action mainly resides in some
interference in melanin formation, regardless of any direct inhibitor/enzyme interaction. Many putative
inhibitors are examined in the presence of tyrosine or dopa as the enzyme substrate, and activity is
assessed in terms of dopachrome formation. Thus, experimental observation of the inhibition of
tyrosinase activity can be accomplished by one of following:
(1) Reducing agents causing chemical reduction of dopaquinone such as ascorbic acid, which is
used as a melanogenesis inhibitor because of its capacity to reduce back o-dopaquinone to dopa, thus
avoiding dopachrome and melanin formations.
(2) o-Dopaquinone scavenger such as most thio-containing compounds, which are well-known
melanogenesis inhibitors and react with dopaquinone to form colorless products. The melanogenetic
process is therefore slowed until all the scavenger is consumed, and then it goes at its original rate.
(3) Alternative enzyme substrates such as some phenolic compounds, whose quinoid reaction
products absorb in a spectral range different from that of dopachrome. When these phenolics show a
good affinity for the enzyme, dopachrome formation is prevented, and they could be mistakenly
classified as inhibitors.
(4) Nonspecific enzyme inactivators such as acids or bases, which non-specifically denature the
enzyme, thus inhibiting its activity.
(5) Specific tyrosinase inactivators such as mechanism-based inhibitors, which are also called
suicide substrates. These inhibitors can be catalyzed by tyrosinase and form covalent bond with the
enzyme, thus irreversibly inactivating the enzyme during catalytic reaction. They inhibit tyrosinase
activity by inducing the enzyme catalyzing suicide reaction.
(6) Specific tyrosinase inhibitors such as most compounds discussed in this review. The compounds
reversibly bind to tyrosinase and reduce its catalytic capacity.
Among the six types of compounds described above, only specific tyrosinase inactivators (5) and
inhibitors (6) are regarded as true inhibitors, which actually bind to the enzyme and inhibit its
activity. General speaking, the mistaken inhibitors exhibit only weak inhibitory activity due to their
reactive and consumable properties toward the enzyme or the quinone products. Although some
tyrosinase inhibitors exhibited multifunctional activities, the compounds, which are well-known
reductive agents, dopaquinone scavengers, or tyrosinase substrates and do not obviously belong to the
true inhibitors, are not suitable for comparison with the true inhibitors and are ignored in the
present article.
Usually, true inhibitors are classified into four types, including competitive inhibitors,
uncompetitive inhibitors, mixed type (competitive/uncompetitive) inhibitors, and non-competitive
inhibitors (Scheme 1). A competitive inhibitor is a substance that combines with a free enzyme in a
manner that prevents substrate binding. That is, the inhibitor and the substrate are mutually exclusive,
Int. J. Mol. Sci. 2009, 10


2445
often because of true competition for the same site. A competitive inhibitor might be a copper chelator,
non-metabolizable analogs, or derivatives of the true substrate. In contrast, an uncompetitive inhibitor
can bind only to the enzyme-substrate complex. A mixed (competitive and uncompetitive mixed) type
inhibitor can bind not only with a free enzyme but also with the enzyme-substrate complex. For most
mixed-type inhibitors, their equilibrium binding constants for the free enzyme and the enzyme-
substrate complex, respectively, are different. However, a special case among the mixed inhibitors is
the non-competitive inhibitors, which bind to a free enzyme and an enzyme-substrate complex with the
same equilibrium constant. In addition to the inhibitory mechanism, inhibitory strength is the primary
criterion of an inhibitor. Inhibitor strength is usually expressed as the inhibitory IC
50
value, which is
the concentration of an inhibitor needed to inhibit half of the enzyme activity in the tested condition.
However, for the tyrosinase inhibitors in the literature, the IC
50
values are incomparable due to the
varied assay conditions, including different substrate concentrations, varied incubation time, and
different batches of commercial tyrosinase. Fortunately, in most studies conducted to discover new
tyrosinase inhibitors, a well-known tyrosinase inhibitor such as kojic acid is often used as a positive
standard at the same time. Hence, in order to compare the inhibitors described in different literature in
a more practical manner, a relative inhibitory activity (RA), which is calculated by dividing the IC
50

value of kojic acid with that of a newly found inhibitor in the same report, is used to express and
compare the inhibitory strength of an inhibitor with others in this review.
Scheme 1. Action mechanism of reversible inhibitors. E, S, I, and P are the enzyme,
substrate, inhibitor, and product, respectively; ES is the enzyme-substrate complex, and EI
and ESI are the enzyme-inhibitor and enzyme-substrate-inhibitor complexes, respectively.

Kojic acid (Figure 3a), the most intensively studied inhibitor of tyrosinase, is a fungal metabolite
currently used as a cosmetic skin-whitening agent and as a food additive for preventing enzymatic
browning [25]. Kojic acid shows a competitive inhibitory effect on monophenolase activity and a
mixed inhibitory effect on the diphenolase activity of mushroom tyrosinase. The ability of kojic acid to
chelate copper at the active site of the enzyme may well explain the observed competitive inhibitory
effect. In addition, kojic acid is reported to be a slow-binding inhibitor of the diphenolase activity of
tyrosinase [26]. This means that the active form of tyrosinase, generated in the catalytic cycle in the
presence of the substrate, is required before binding of the inhibitor to the enzyme can occur. Other
E + S ES E + P
K
1

+
I
EI
+
I
ESI
K
3

K
2
K
4

Competitive Uncompetitive
Mixed or Non-competitive (as K
2
= K
4
)
Int. J. Mol. Sci. 2009, 10


2446
slow-binding inhibitors of tyrosinase are the very potent inhibitor tropolone [27] and the substrate
analog L-mimosine [28] (Figure 3a). Strikingly, these slow-binding inhibitors of tyrosinase all contain
an -hydroxyketone group. Kojic acid together with tropolone and L-mimosine are often used as the
positive control in the literature for comparing the inhibitory strength of the finding inhibitors.
In addition to the standard tyrosinase inhibitors, a huge number of new inhibitors, especially for
those discovered in the last five years, are collected in this review and classified into five major classes,
including polyphenols, benzaldehyde and benzoate derivatives, long-chain lipids and steroids, other
natural or synthetic inhibitors, and irreversible inactivators based on either the chemical structures or
the inhibitory mechanism.
3.1. Polyphenols
Polyphenols represent a diverse group of compounds containing multiple phenolic functionalities
and are widely distributed in nature. Polyphenols are also the largest groups in tyrosinase inhibitors
until now. Since several polyphenols are accepted as substrates by tyrosinase, it depends on the
presence and position of additional subsistent whether a polyphenol may act as an inhibitor.
Flavonoids are among the most numerous and best-studied polyphenols, that is, benzo--pyrone
derivatives consisting of phenolic and pyrene rings. Widely distributed in the leaves, seeds, bark, and
flowers of plants, more than 4,000 flavonoids have been identified to date. In plants, these compounds
give protection against UV radiation, pathogens, and herbivores [29]. They are also responsible for the
characteristic red and blue colors of berries, wines, and certain vegetables. Flavonoids may be
subdivided into seven major groups, including flavones, flavonols, flavanones, flavanols, isoflavonoids,
chalcones, and catechin. Different classes of flavonoids are distinguished by additional oxygen-
heterocyclic rings, by positional differences of the B ring, and by hydroxyl, methyl, isoprenoid, and
methoxy groups distributed in different patterns about the rings. The structure of flavonoids is also in
principle compatible with the roles of both substrates and (presumably competitive) inhibitors of
tyrosinase. Detailed studies have shown that some flavonoids are in fact rather potent inhibitors and
discussed in this section. In addition to flavonoids, other polyphenols, which were also identified as
tyrosinase inhibitors, contain stilbenes and coumarin derivatives.

Flavonols. Many flavonols have been isolated from plants, and some were identified as tyrosinase
inhibitors. The inhibitory mode of flavonol inhibitors is usually competitive inhibition for the
oxidation of L-dopa by tyrosinase and the 3-hydroxy-4-keto moiety of the flavonol structure acts as the
key role in copper chelation [30-31]. In terms of inhibitor strength, the discovered flavonol inhibitors
are ranking as follows: quercetin (5,7,3',4'-tetrahydroxyflavonol) > myricetin (5,7,3',4',5'-
pentahydroxy-flavonol) > kaempferol (5,7,4'-trihydroxyflavonol) > galangin (5,7-
dihydroxyflavonol) >> morin, buddlenoid A, buddlenoid B [32-33]. In addition, the flavonol
glycosides of quercetin or kaempferol were found to be less active than their corresponding aglycones
[34]. Recently, 6-hydroxykaempferol was synthesized and confirmed to possess two times more
activity than kaempferol [35]. Although many flavonols have been identified as tyrosinase inhibitors,
all the flavonol inhibitors listed above are very weak inhibitors; the most active flavonol, quercetin
(Figure 3b), showed only 20% of the inhibitory strength of kojic acid toward diphenolase activity of
Int. J. Mol. Sci. 2009, 10


2447
mushroom tyrosinase. Hence, it is obvious that these flavonol inhibitors have little potential in
applications of skin whitening or food antibrowning.
Figure 3. Chemical structures of selected tyrosinase inhibitors belonging to some standard
ones (a), flavonoids (b-j) or N-benzylbenzamides analogs (k). RA
a
and RA
b
are the relative
diphenolase and monophenolase inhibitory activity, respectively, against mushroom
tyrosinase compared to the standard kojic acid, where 1.0F means one time activity of
kojic acid.
O
OH
N
O
O H
OH
O
NH
2
O
OH
O H
OH
O
O H
O
OH
O H
OH
OH
OH
O
O
OH
OH
O
O H
O H
OH
O
OH
OH
O
O H
OH
OH
O
OH
O H
O
(a) Standard tyrosinase inhibitors
Kojic acid Tropolone L-Minosine
(b) Flavonols
Quercetin
(Competitive; RA
a
, 0.2F; ref.32)
(c) Flavones
(d) Flavanones
Norartocarpetin: R=OH
(Competitive; RA
b
, 10.4F; ref.39)
Artocarpetin: R=OCH
3
(RA
b
< 0.1F; ref.43)
Streppogenin
(Competitive; RA
b
, 13.6F; ref.41)
(e) Flavanols
Dihydromorin
(RA
b
, 0.5F; ref.43)
Taxifolin
(RA
b
, 1.0F; ref.46)
O O
OH
OH
O
OH
R2
R3
R1 O
O H O
O
OH
OMe
O H
(g) Isoflavans
Glabridine
(Non-competitive; RA
b
, 15.2F; ref.51)
Glyasperin C
(RA
b
, 27.7F; ref.51)
O O H
OH
OH
OH
(h) Isoflavones
Calycosin
(RA
b
, 1.3F; ref.57)
6-Hydroxydaidzein: R1=R3=H, R2=OH
(Competitive; RA
b
, 6.0F; ref.53)
8-Hydroxydaidzein: R1=R2=H, R3=OH
(Suicide substrate; ref.54)
8-Hydroxygenistein: R1=R3=OH, R2=H
(Suicide substrate; ref.54)
(f) Isoflav-3-en
O
OH
OH
O
O H
R
Haginin A
(Non-competitive; RA
b
, 10.1F; ref.55)
O
OH
O H
OMe
OMe
(i) Chalcones
2,4,2',4'-Tetrahydroxychalcone: R=H
(Competitive; RA
b
, 2.5F; ref.66)
2,4,6,2',4'-Pentahydroxychalcone: R=OH
(Competitive; RA
b
, 12.0F; ref.66)
R O H
O
OH O H
OH
(j) Prenylated Chalcones
O H
O
OH MeO
Licochalcone A
(Competitive; RA
b
, 5.4F; ref.58)
O H
O
OH O H
OH
TMBC
(Competitive; RA
b
, 26.1F; ref.61)
O H
O
OH O H
OMe
OH O H
O
OH O H
OMe
OH
OH
Kuraridin
(RA
b
, 34.1F; ref.59)
Kuraridinol
(Non-competitive; RA
b
, 18.4F; ref.60)
(k) N-Benzylbenzamides
N
H
O
R1
R2
R3
R4
R5
OH
3,5,2',4'-Tetrahydroxyl derivatives: R1=R3=H,
R2=R4=R5=OH (RA
a
, 7.4F; ref.67)
2,4,2',4'-Tetrahydroxyl derivatives: R1=R3=R5=OH,
R2=R4=H (RA
a
, 0.6F; ref.67)
3,5,4'-Trihydroxyl derivatives: R1=R3=R5=H,
R2=R4=OH (RA
a
<<0.1F; ref.67)
2,4,4'-Trihydroxyl derivatives: R1=R3=OH,
R2=R4=R5=H (RA
a
<<0.1F; ref.67)
O
OH
N
O
O H
OH
O
NH
2
O
OH
O H
OH
O
O H
O
OH
O H
OH
OH
OH
O
O
OH
OH
O
O H
O H
OH
O
OH
OH
O
O H
OH
OH
O
OH
O H
O
(a) Standard tyrosinase inhibitors
Kojic acid Tropolone L-Minosine
(b) Flavonols
Quercetin
(Competitive; RA
a
, 0.2F; ref.32)
(c) Flavones
(d) Flavanones
Norartocarpetin: R=OH
(Competitive; RA
b
, 10.4F; ref.39)
Artocarpetin: R=OCH
3
(RA
b
< 0.1F; ref.43)
Streppogenin
(Competitive; RA
b
, 13.6F; ref.41)
(e) Flavanols
Dihydromorin
(RA
b
, 0.5F; ref.43)
Taxifolin
(RA
b
, 1.0F; ref.46)
O O
OH
OH
O
OH
R2
R3
R1 O
O H O
O
OH
OMe
O H
(g) Isoflavans
Glabridine
(Non-competitive; RA
b
, 15.2F; ref.51)
Glyasperin C
(RA
b
, 27.7F; ref.51)
O O H
OH
OH
OH
(h) Isoflavones
Calycosin
(RA
b
, 1.3F; ref.57)
6-Hydroxydaidzein: R1=R3=H, R2=OH
(Competitive; RA
b
, 6.0F; ref.53)
8-Hydroxydaidzein: R1=R2=H, R3=OH
(Suicide substrate; ref.54)
8-Hydroxygenistein: R1=R3=OH, R2=H
(Suicide substrate; ref.54)
(f) Isoflav-3-en
O
OH
OH
O
O H
R
Haginin A
(Non-competitive; RA
b
, 10.1F; ref.55)
O
OH
O H
OMe
OMe
(i) Chalcones
2,4,2',4'-Tetrahydroxychalcone: R=H
(Competitive; RA
b
, 2.5F; ref.66)
2,4,6,2',4'-Pentahydroxychalcone: R=OH
(Competitive; RA
b
, 12.0F; ref.66)
R O H
O
OH O H
OH
(j) Prenylated Chalcones
O H
O
OH MeO
Licochalcone A
(Competitive; RA
b
, 5.4F; ref.58)
O H
O
OH O H
OH
TMBC
(Competitive; RA
b
, 26.1F; ref.61)
O H
O
OH O H
OMe
OH O H
O
OH O H
OMe
OH
OH
Kuraridin
(RA
b
, 34.1F; ref.59)
Kuraridinol
(Non-competitive; RA
b
, 18.4F; ref.60)
(k) N-Benzylbenzamides
N
H
O
R1
R2
R3
R4
R5
OH
3,5,2',4'-Tetrahydroxyl derivatives: R1=R3=H,
R2=R4=R5=OH (RA
a
, 7.4F; ref.67)
2,4,2',4'-Tetrahydroxyl derivatives: R1=R3=R5=OH,
R2=R4=H (RA
a
, 0.6F; ref.67)
3,5,4'-Trihydroxyl derivatives: R1=R3=R5=H,
R2=R4=OH (RA
a
<<0.1F; ref.67)
2,4,4'-Trihydroxyl derivatives: R1=R3=OH,
R2=R4=R5=H (RA
a
<<0.1F; ref.67)


Int. J. Mol. Sci. 2009, 10


2448
Flavones, flavanones, and flavanols. Citrus peel as a by-product of the citrus juice industry contains a
large amount of flavonoids. Some of the flavonoids were identified as tyrosinase inhibitors, including
nobiletin (5,6,7,8,3',4'-hexamethoxyflavone), naringin (5,7,4'-trihydroxyflavanone), and neohesperidin
(5,7,3'-trihydroxy-4'-methoxyflavone). However, the inhibitory strength of the three inhibitors was
found to be poorly active toward mushroom tyrosinase compared with kojic acid [36-37]. Although no
potent tyrosinase inhibitors have been isolated from citrus fruit until now, the ethanolic extract of
citrus fruit exhibited in vitro inhibitory effects on melanogenesis in melanoma cells and in vivo
prevention against UVB-induced pigmentation of dorsal skin in brown guinea pigs. The melanogenesis
inhibitory activity of citrus crude extracts was found to be mainly attributed to the antioxidant activity
of neohesperidin in citrus fruit.

In addition to citrus extracts, the extracts from Morus species, which has been well-known as a
polyphenol-rich plant and used as a non-toxic natural therapeutic agent, also have high potential in
applications as skin-whitening agents due to many potent tyrosinase inhibitors being isolated from
different parts of the plant. Mulberroside F (moracin M-6,3'-di-O--glucopyranoside) purified from the
leaves of the plant showed the antidiphenolase activity of mushroom tyrosinase to be 4.5-fold higher
than that of kojic acid and exhibited an inhibitory effect on melanin formation within melanoma cells
[38]. Norartocarpetin (5,7,2',4'-tetrahydroxyflavone, Figure 3c), isolated from the stem bark of the
plant, was found to be 10.4-fold more active than kojic acid against monophenolase activity of
mushroom tyrosinase with a competitive inhibition mode (K
I
= 1.35 M) [39]. The flavone was also
demonstrated to be a slow-binding inhibitor just like kojic acid and tropolone. In addition to the leaves
and stem of the plant, the roots of the Morus species were also found to contain many very potent
tyrosinase inhibitors, including oxyresveratrol [40], norartocarpetin, and streppogenin (5,7,2',4'-
tetrahydroxy-flavavone, Figure 3d) [41]. Oxyresveratrol is a hydroxystilbene with trans configuration,
and its inhibitory property will be discussed in the later silbenes section. The chemical structure of
streppogenin (a flavanone) is very similar to that of norartocarpetin (a flavone) with the same four
substituted hydroxyl groups. It is reasonable that streppogenin possesses an inhibitory property similar
to that of norartocarpetin including similarly inhibitory strength on monophenolase activity
(IC
50
= 0.57 and 0.47 M, respectively) and the same slow-binding and competitive inhibition mode
(K
I
= 0.7 and 0.6 M, respectively) against mushroom tyrosinase. Moreover, the two potent inhibitors,
together with another two analogs, dihydromorin (5,7,2',4'-tetrahydroxyflavanol, Figure 3e) and
artocarpetin (5,2',4'-trihydroxy-7-methoxyflavone, Figure 3c), were recently isolated from the wood of
Artocarpus heterophyllus [42-43].
It is very interesting to compare the inhibitory strength toward monophenolase activity of
mushroom tyrosinase among the four analogs described above. First, it was found that methoxylation
of the hydroxyl group at the C7 position of the flavone skeleton revealed a 100-fold decrease in
inhibitory activity by comparing the inhibitory strength between artocarpetin and norartocarpetin. In
fact, for most tyrosinase inhibitors, glycosylation and methoxylation of the specific hydroxyl group on
the aromatic ring would heavily affect exerting their inhibitory activity. Similar results were also found
that forty-five flavonoids glycosides were recently isolated from Marrubium species, and all exhibited
very low tyrosinase inhibitory activity [44]. The reasons for causing the decreased inhibitory activity
by glycosylation and methoxylation of flavonoids include the loss of the specific hydroxyl group,
Int. J. Mol. Sci. 2009, 10


2449
which plays a key role in showing the inhibition and formation of the hydrophilic and stereohindrance
effects of the bulky glycoside moiety, which prevent the inhibitor from fitting into the active site of the
enzyme. Second, it has been mentioned that norartocarpetin and streppogenin have very similar
inhibitory activity and mode toward mushroom tyrosinase. In contrast, dihydromorin, which contains
an extra 3-hydroxy group and hence belongs to the flavanol class of flavonoids, exhibited a 20-fold
decrease in inhibitory activity compared with that of streppogenin and norartocarpetin. As described in
the flavonol section, it has been demonstrated that the 3-hydroxy-4-keto moiety in the flavonol
inhibitors acts as the key role in copper chelation, and loss of the group will completely abolish
inhibitory activity [31]. Based on the mechanism, it is difficult to explain the lowered inhibitory
capacity of dihydromorin, which contains the 3-hydroxy group and thus forms the 3-hydroxy-4-keto
moiety in its structure. Although the 3-hydroxy-4-keto moiety of dihydromorin could form the copper
chelating capacity, it seems that the extra 3-hydroxy group in dihydromorin had more drawbacks on
tyrosinase inhibition. According to this, the criteria for tyrosinase inhibition by the flavonoids become
an interesting issue. Especially, the effects of both the numbers and the positions of hydroxyl groups
attached to the flavonoids skeletons on the inhibitory activity of mushroom tyrosinase are the main
concern. Kim et al. recently systematically tested a large number of flavonoids with different numbers
and positions of hydroxyl groups using the fluorescence quenching spectroscopic method [45]. Both
the tyrosinase inhibitory activities and copper chelating capacities of the flavonoids were evaluated in
the work. From the results, the authors concluded that the enzyme tyrosinase is primarily quenched by
the hydroxyl groups of A and B rings on the ether side of the flavonoids (C6 to C8 and C2 to C4).
According to the finding, the extra 3-hydroxyl group of dihydromorin would indeed reduce its
inhibitory activity compared to streppogenin due to the 3-hydroxyl group making dihydromorin more
difficult to form a binding complex with the enzyme. Their result could also explain that most
flavonols or flavanols inhibitors exhibited only weak to moderate inhibitory strength.
Another flavanol, taxifolin (5,7,3',4'-tetrahydroxyflavanol, Figure 3e) isolated from the sprout of
Polygonum hydropiper, showed equal inhibitory activity of kojic acid toward monophenolase activity
of mushroom tyrosinase [46]. In an advance study, it is interesting to find that taxifolin would
effectively inhibit both the tyrosinase activity of the living cells and cellular melanogenesis as
effectively as arbutin, despite its effects on increasing the tyrosinase protein level [47]. In addition to
flavonoid monomers, one flavone-flavanone dimmer was isolated from seashore plants, Garcinia
subelliptica, and revealed to be 3.6-fold more active than kojic acid against the monophenolase
activity of mushroom tyrosinase [48].

Isoflavonoids. The extracts from the roots and seeds of Glycyrrhiza species (Leguminoseae) have long
been regarded as an effective constituent for skin-whitening agents in East Asian countries. The
melanogenesis inhibitory activity of the extracts mainly comes from the isoflavonoids in the plant.
Two isoflavans were purified from the roots of the plant and identified as potent tyrosinase inhibitors.
Glabridine (Figure 3g) was the first confirmed inhibitor with 15 times activity of kojic acid and
exhibited higher depigmenting activity than that of arbutin [49]. In contrast, glabridines analog,
glabrene, was found to be 100-fold less active than glabridine [50]. Kinetics study showed that
glabridin and glabrene inhibited the enzyme with non-competitive and uncompetitive modes,
respectively. Glyasperin C (Figure 3g) was recently isolated from the same part of the plant and shown
Int. J. Mol. Sci. 2009, 10


2450
to be two times more active than glabridin [51]. Although glyasperin C is the most active inhibitor,
glabridin had the best melanogenesis inhibitory activity among them.

On the other hand, we isolated several isoflavones derivatives from soybean koji fermented with
Aspergillus oryzae and demonstrated three hydroxyisoflavones including 6-hydroxydaidzein (6,7,4'-
trihydroxyisoflavone), 8-hydroxydaidzein (7,8,4'-trihydroxyisoflavone), and 8-hydroxygenistein
(5,7,8,4'-tetrahydroxyisoflavone) as potent tyrosinase inhibitors (Figure 3h) [52-53]. Among them,
6-hydroxydaidzein with 6-fold more than kojic acid acts competitively on the L-tyrosine binding site
of the enzyme while the other two 8-hyroxyisoflavones irreversibly inactivate the enzyme and belong
to the suicide substrates of tyrosinase (discussed in the irreversible inactivators section) [54]. It is
interesting to note that the position and number of hydroxyl groups in the A ring of the isoflavone
structure can strongly affect both the inhibitory strength and the inhibitory mode of the isoflavones to
mushroom tyrosinase. For example, an isoflavone with hydroxyl groups at both the C6 and C7
positions in the A ring (6,7,4'-trihydroxyisoflavone) would increase more than 10 times both the
inhibitory activity (judged by IC
50
values) and affinity to the enzyme (judged by Michaelis constants)
compared to that of isoflavones either with only one hydroxyl group at the C7 position or without any
hydroxyl group in the A ring. Alternatively, the hydroxyl groups at both the C7 and C8 positions
(7,8,4'-trihydroxyisoflavone and 5,7,8,4'-tetrahydroxyisoflavone) would completely change the
inhibitory mode of the isoflavones from the reversible competitive to the irreversible suicide form. The
elucidation of the detailed mechanism of the effects of the hydroxyl groups in the A ring in the
isoflavones structure on their inhibitory activity toward tyrosinase needs further study.
In addition to the isoflavonoids from Glycyrrhiza plants, Lee et al. recently identified other natural
isoflavonoids as potent tyrosinase inhibitors. Haginin A (2',3'-dimethoxy-7,4'-dihdroxyisoflav-3-ene,
Figure 3f) isolated from the branch of Lespedeza cyrtobotrya was shown to be 10-fold more active
than kojic acid against monophenolase activity of mushroom tyrosinase with a non-competitive mode
[55]. The compound significantly inhibited melanin synthesis in melanoma cells and decreased UV-
induced skin pigmentation in brown guinea pigs. Furthermore, haginin A presented remarkable
inhibition on the body pigmentation in the zebrafish model system. Dalbergioidin (5,7,2',4'-
tetrahyroxyisoflavan) was also isolated from L. cyrtobotrya and non-competitively inhibited the
monophenolase activity of mushroom tyrosinase [56]. Fifty percent of melanin biosynthesis in
melanoma cells was inhibited by 27 M of dalbergioidin, and 80% of cell viability was maintained in
this concentration. The third potent inhibitor discovered by the authors is calycosin (4'-methoxy-7,4'-
dihydroxyisoflavone, Figure 3h), which showed slighter higher monophenolase inhibitory activity
toward mushroom tyrosinase than that of kojic acid. The compound contains two mechanisms to
reduce melanogenesis in melanoma cells, including inhibiting tyrosinase activity and reducing the
expression of tyrosinase [57].

Chalcones. Chalcones consist of two aromatic rings in trans configuration, separated by three carbon
atoms, of which two are connected by a double bond and the third is a carbonyl group. Some natural
prenylated chalcones showed potent tyrosinase inhibitory activity. Three chalcones derivatives,
including licuraside, isoliquiritin, and licochalcone A were isolated from the roots of the Glycyrrhiza
species and competitively inhibited the monophenolase activity of mushroom tyrosinase. Among them,
Int. J. Mol. Sci. 2009, 10


2451
licochalcone A (Figure 3j) was 5.4-fold more active than kojic acid [58]. Another prenylated chalcone,
kuraridin (Figure 3j), was isolated from the plant Sophora flavescens and identified as a potent
tyrosinase inhibitor, which was 34 times activity of kojic acid against monophenolase activity of
mushroom tyrosinase [59]. Following, its hydroxyl analog, kuraridinol (Figure 3j), was found to be
18.4-fold more active compared to kojic acid [60]. More interesting, the two prenylated chalcones
exhibited significantly more activity than those of their corresponding flavanone analogs. Kuraridin (a
chalcone) is 10-fold more active than kurarinone (a flavanone) while kuraridinol (a chalcone) is also
10-fold more active than kurarinol (a flavavone). This emphasizes that tyrosinase inhibitors with a
chalcone structure have enhanced potential. Recently, 2,4,2',4'-tetrahydroxy-3-(3-methyl-2-butenyl)-
chalcone (TMBC, Figure 3j), which was isolated from the stems of Morus nigra, was proved to be
26-fold more potent than kojic acid in diphenolase inhibitory activity of mushroom tyrosinase [61].
The kinetic study showed that the compound is competitive to the L-dopa binding site of the enzyme
with a K
I
value of 1 to 1.5 M. Moreover, the melanin content of melanoma cells was reduced to 31%
by 30 M of TMBC treatment. The inhibitory effect of TMBC on melanogenesis was attributed to the
direct inhibition of tyrosinase activity, rather than suppression of tyrosinase gene expression.

From the naturally found chalcones listed above, it seems that the 4-resorcinol moiety (2,4-
dihydroxyl groups in the aromatic ring) in the chalcone structure is the key substituted group in
exerting potent inhibitory activity. Some simple 4-alkylresorcinols were proved to exhibit strong
tyrosinase inhibitory activity [62-63]. To evaluate the structure-activity relationship between the
numbers and the positions of hydroxyl groups in the chalcone skeleton and the inhibitory activity
toward mushroom tyrosinase, the inhibitory activity against mushroom tyrosinase of many chemically
synthesized hydroxychalcones was examined. In an early study, Nerya et al. reported that the
4-hydroxyl group in the B ring of the chalcone skeleton is the major factor affecting inhibitory potency,
because it results in a molecular skeleton closely similar to that of L-tyrosine [64]. Following, they
found that 2,4,2',4'-tetrahydroxychalcone (Figure 3i) possesses the most potent monophenolase
inhibitory activity compared with 3,4,2',4'-tetrahydroxychalcone and 2,4,3',4'-tetrahydroxychalcone
[65]. Therefore, they concluded that 4-resorcinol moiety in the chalcone skeleton plays the key role in
exhibiting inhibitory potency. Similarly, Jun et al. recently chemically synthesized a series of
hydroxychalcones and determined the tyrosinase inhibitory activity [66]. The results were also
consistent with that by Nerya et al. Moreover, they found that 2,4,2',4',6'-pentahydroxychalcone
(Figure 3i) is 5-fold stronger than 2,4,2',4'-tetrahydroxychalcone. The inhibitory mode of the
compound is also competitive to the L-tyrosine binding site of the enzyme with a K
I
value of 3.1 M.
On the other hand, as seen in the description in the earlier flavone section, it is interesting to note
that the identified potent tyrosinase inhibitors with a flavone, flavanone, or flavonol skeleton such as
artocarpetin, norartocarpetin, and streppogenin all contain 4-resorcinol in the B ring. Thus, the
4-resorcinol moiety plays an important role in the inhibition of tyrosinase activity not only in
chalcones but also in other flavonoid structures.
In similar research with N-benzylbenzamide as the structure skeleton, which is analogous to that of
chalcone but with an amide moiety (not the alkyl double bond) connecting between the two aromatic
rings, the inhibitory activities of N-benzylbenzamide derivatives (Figure 3k) were found to rank by
3,5,2',4'-tetrahydroxyl > 2,4,2',4'-tetrahydroxyl > 3,5,4'-trihydroxyl > 2,4,4'-trihydroxyl substitutions
Int. J. Mol. Sci. 2009, 10


2452
[67]. Accordingly, it was concluded that the 4-resorcinol in the B ring has more effect than in the A
ring on exerting potent inhibitory activity, while the 5-resorcinol in the A ring is preferred to the 4-
resorcinol. Very interesting, the structural criterion for exerting potent tyrosinase inhibitory activity in
the chalcone-like structure, i.e., the 4-resorcinol moiety in the B ring and the 5-resorcinol in the A ring,
is partially consistent with that in the flavone structure studied by the previous fluorescence quenching
spectroscopic method, i.e., the hydroxyl groups of A and B rings on the ether side of the flavones
(C6 to C8 and C2 to C4) [45]. In addition, Khatib et al. recently used computer modeling to dock the
enzyme crystallographic structure by some 4-resorcinol derivatives with different ester chains and
compared the experimental inhibitory IC
50
values with the calculated free energy and docking energy
[68]. The authors results emphasize again the importance of the 4-resorcinol skeleton in exerting
potent tyrosinase inhibitory activity and the extent to which a lipophilic moiety, combined with the
resorcinol skeleton, can contribute to this activity. The additional lipophilic groups affect both the
inhibition potency, as well as the ability of the tyrosine to compete with the inhibitors. Such a
lipophilic unit, which contains a minor bulky group, was a preferred inhibitor over a long chain or
highly bulky functional moiety, as it may interact with the enzyme hydrophobic pocket and augment
binding affinity. The conclusion could explain the potent inhibitory activity of prenylated chalcone
with 4-resorcinol moiety such as kuraridin and kuraridinol described above.

Stilbenes. A stilbene consists of an ethene double bond substituted with a benzyl ring on both carbon
atoms of the double bond. Oxyresveratrol (2,4,3',5'-tetrahydroxy-trans-stilbene, Figure 4a), which was
initially isolated from Morus alba, exhibited 32-fold more inhibitory activity than that of kojic acid
[40]. The inhibitor acts non-competitively on both monophenolase and diphenolase activity of
mushroom tyrosinase. Based on the potent inhibitory capacity, oxyresveratrol reduced pigmentation in
melanoma cells. The melanogenesis inhibitory mechanism of the compound was proved to directly
inhibit enzyme activity but not affect gene expression [69]. It is worth noting that oxyresveratrol also
contains the structure of the 4-resorcinol moiety in the B ring and the 5-resorcinol moiety in the A ring
as described in the former chalcone section. In fact, oxyresveratrol is 50-fold more active than its
analog, resveratrol (2,3',5'-trihydroxy-trans-stilbene), which loses the 4-resorcinol moiety in its
structure. Taken together with all these findings, the key role of the structural criterion (the
4-resorcinol moiety in the B ring and the 5-resorcinol moiety in the A ring) of an inhibitor in
tyrosinase inhibition has been validated in flavones, chalcones, and stilbenes, and this criterion
becomes a golden rule for further design of a potent tyrosinase inhibitor.

Following oxyresveratrol, another three hydroxystilbenes were also purified and identified as potent
tyrosinase inhibitors. Chloroporin (4-geranyl-3,5,2',4'-tetrahydroxy-trans-stilbene, Figure 4a) was
purified from the heartwood of Chlorophora excelsa and displayed 14.8-fold inhibitory activity of
kojic acid against diphenolase of mushroom tyrosinase [70]. Gnetol (2,6,3',5'-tetrahydroxy-trans-
stilbene, Figure 4a), isolated from the roots of Gnetum gnemon, exhibited 30-fold more diphenolase
inhibitory activity of murine tyrosinase than that of kojic acid [71]. Recently, piceatannol (3,5,3',4'-
tetrahydroxy-trans-stilbene, Figure 4a), isolated from grapes and red wine, showed 32.7-fold
antimonophenolase activity of kojic acid toward mushroom tyrosinase [72]. The hydroxyl positions of
the structure of piceatannol, which contains o-(3',4'-)dihydroxy groups, are significantly different from
Int. J. Mol. Sci. 2009, 10


2453
other naturally found hydroxystilbenes. The o-dihydroxy groups play an important role in antioxidant
activity (free radical scavenging). In fact, piceatannol has strong suppressive activity in reactive
species generation and enhances the reduced/oxidized glutathione ratio in cells. Hence, the inhibitory
mechanism of piceatannol on tyrosinase monophenolase activity may be exerted by both direct
tyrosinase inhibition and quinone products scavenging, and is not the same with that of oxyresveratrol.
However, a detailed kinetic study of the inhibition by piceatannol is lacking.
Aside from naturally occurring hydroxystilbenes, a number of synthetic stilbene derivatives are also
good tyrosinase inhibitors. To examine the structure-activity relationship, a variety of hydroxystilbene
compounds were synthesized and assayed for their tyrosinase inhibitory activity. The role of the
double bond between the two aromatic rings of the stilbene skeleton in the tyrosinase inhibition has
been discussed in these studies. By comparing the diphenolase inhibitory activity of murine tyrosinase
by cis- and trans-isomers of 3,3'-dihydroxystilbene, it was concluded that the trans-olefin structure of
the parent stilbene skeleton is essential for inhibition [73]. Indeed, all the natural hydroxystilbenes
possessing potent antityrosinase activity are trans-isomers. However, recently, Song et al., concluded
the opposite: they found that the diphenolase inhibitory capacity on mushroom tyrosinase by cis-3,5-
dihydroxystilbene was stronger than that by its corresponding trans-isomer [74]. The reasons to
explain the obtainted opposite results are not clear. On the other hand, a gnetol analog, dihydrognetol,
which loses the double bond between the two aromatic rings, exerted a greatly lowered inhibitory
effect on diphenolase activity of murine tyrosinase [73], and it was concluded that the role of the
double bond in the stilbene skeleton for tyrosinase inhibition is critical. In contrast, an oxyresveratrol
analog, dihydrooxyresveratrol (Figure 4b), which also loses the double bond between the two aromatic
rings, showed 8-fold more diphenolase inhibitory activity toward mushroom tyrosinase than its analog,
oxyresveratrol [75]. The authors suggested that the higher tyrosinase inhibitory activity of
dihydrooxyresveratrol was probably due to its bibenzyl structure, which gave more flexibility and thus
allowed the phenolic groups to interact with the enzyme more effectively. Similar results were also
found by both Oozeki et al. [76] and Vielhaber et al. [77]. The former authors synthesized another
bibenzyl analog but with 2,4,2',4'-tetrahydroxyl substitution groups (Figure 4b) and identified the
compound to be 20-fold more active than kojic acid while the latter authors found that 4-(1-
phenylethyl)1,3-benzenediol (Figure 4b) inhibited mushroom tyrosinase 22 times more effective than
kojic acid. These results implied again that the double bond in the stilbene structure is not essential.
Despite the undefined inhibitory mechanism of hydroxystilbenes, one Korean research group led by
Chung and Suh recently successfully synthesized a new isostere of oxyresveratrol, HNB [4-(6-
hydroxy-2-naphthyl)-1,3-bezendiol, Figure 4b], which exhibited 546-fold more inhibitory activity
toward monophenolase of mushroom tyrosinase than that of kojic acid [78]. HNB is also the strongest
tyrosinase inhibitor published until now. The kinetic study demonstrated that HNB is a competitive
inhibitor with a K
I
value of 4.78 to 6.21 nM. Due to the potent tyrosinase inhibitory activity, it is
reasoned that HNB suppressed cellular tyrosinase activity and total melanin content to 27% and 35%,
respectively, in melanoma cells at 100 M concentration, where cells maintained 87% viability [79].

Coumarins. Coumarins are lactones of phenylpropanoid acid with an H-benzopyranone nucleus.
Among the coumarin-type tyrosinase inhibitors, aloesin (Figure 4c) is the famous one, which is a
natural hydroxycoumarin glucoside isolated from Aloe vera. Aloesin performed more inhibitory
Int. J. Mol. Sci. 2009, 10


2454
activity toward crude murine tyrosinase than mushroom tyrosinase and has been recently used in
topically applied cosmetics due to its natural source and multifunctional activity in skin care [80-81].
A coumarin analog, esculetin, was isolated from the seeds of Euphorbia lathyris and showed one-
quarter of the antityrosinase activity of kojic acid [82]. However, the compound was recently
demonstrated to be a substrate of mushroom tyrosinase [83]. Another coumarin analog, 9-hydroxy-4-
methoxypsoralen (Figure 4c), was isolated from Angelica dahurica and exhibited six times more
tyrosinase inhibitory activity than that of kojic acid [84]. Recently, a new coumarin derivative, 8'-epi-
cleomiscosin A (Figure 4c), was isolated from the aerial parts of Rhododendron collettianum and
showed 12.8-fold diphenolase inhibitory activity of kojic acid toward mushroom tyrosinase [85].
Interestingly, another purified compound, cleomiscosin A, had a structure highly similar to that of
8'-epi-cleomiscosin A but exhibited a 14-fold decrease in tyrosinase inhibitory activity compared to
that of 8'-epi-cleomiscosin A. The only difference between these two compounds is that the -proton
at the position 8' for the former, where the -proton at the same position for the latter. A change in
stereochemistry of a single proton that drastically changes the inhibitory activity of the compound is
rarely found in the reviewed literature.
Figure 4. Chemical structures of selected tyrosinase inhibitors belonging to stilbenes (a),
bibenzyl derivatives (b), coumarins (c), and benzaldehyde derivatives (d). RA
a
and RA
b

have the same meanings as those of Figure 3.
(a) Stilbenes
(b) Bibenzyl derivatives
2,4,3',5'-Tetrahydroxybibenzyl:
R1=R3=H, R2=R4=OH
(RA
a
, 83.4F; ref.75)
2,4,2',4'-Tetrahydroxybibenzyl:
R1=R3=OH, R2=R4=H
(RA
a
, 20.1F; ref.76)
2,4,2'-Trihydroxybibenzyl:
R1=OH, R2=R3=R4=H
(RA
a
, 22.3F; ref.77)
Chloroporin
(RA
b
, 14.8F; ref.70)
O H
O H
R1
R4
R3
R2
Oxyresveratrol: R1=R3=OH, R2=R4=H
(Non-competitive; RA
b
, 33.4F; ref.69)
Gnetol: R1=R4=OH, R2=R3=H
(RA
b
, 29.2F; ref.71)
Piceatannol: R1=R4=H, R2=R3=OH
(RA
b
, 32.7F; ref.72)
O H
O H
O H
OH
R2
R4
O H
OH
R1
R3
OH
OH
O H
HNB
(Competitive, RA
b
, 546F; ref.79)
(c) Coumarins
(d) Benzaldehyde derivatives
O H
O H
O
3,4-Dihydroxybenzaldehyde-O-ethyloxime
(RA
a
, 73.1F; ref.106)
Protocatechualdehyde
(Competitive; RA
b
, 7.8F; ref.100)
O H
O H
N
O
O
O
O
OH
OMe
9-Hydroxy-4-methoxypsoralen
(Non-competitive; RA
b
, 6.4F; ref.84)
O
O
O
R2
R1
H
O H
O
OMe
MeO
8'-epi-cleomiscosin A: R1=H, R2=CH
2
OH
(RA
a
, 12.8F; ref.85)
Cleomiscosin A: R1=CH
2
OH, R2=H
(RA
a
, 0.8F; ref.85)
O O H
Clu
O
O
Aloesin
(Competitive; RA
b
, 0.2F; ref.80)
(a) Stilbenes
(b) Bibenzyl derivatives
2,4,3',5'-Tetrahydroxybibenzyl:
R1=R3=H, R2=R4=OH
(RA
a
, 83.4F; ref.75)
2,4,2',4'-Tetrahydroxybibenzyl:
R1=R3=OH, R2=R4=H
(RA
a
, 20.1F; ref.76)
2,4,2'-Trihydroxybibenzyl:
R1=OH, R2=R3=R4=H
(RA
a
, 22.3F; ref.77)
Chloroporin
(RA
b
, 14.8F; ref.70)
O H
O H
R1
R4
R3
R2
Oxyresveratrol: R1=R3=OH, R2=R4=H
(Non-competitive; RA
b
, 33.4F; ref.69)
Gnetol: R1=R4=OH, R2=R3=H
(RA
b
, 29.2F; ref.71)
Piceatannol: R1=R4=H, R2=R3=OH
(RA
b
, 32.7F; ref.72)
O H
O H
O H
OH
R2
R4
O H
OH
R1
R3
OH
OH
O H
HNB
(Competitive, RA
b
, 546F; ref.79)
(c) Coumarins
(d) Benzaldehyde derivatives
O H
O H
O
3,4-Dihydroxybenzaldehyde-O-ethyloxime
(RA
a
, 73.1F; ref.106)
Protocatechualdehyde
(Competitive; RA
b
, 7.8F; ref.100)
O H
O H
N
O
O
O
O
OH
OMe
9-Hydroxy-4-methoxypsoralen
(Non-competitive; RA
b
, 6.4F; ref.84)
O
O
O
R2
R1
H
O H
O
OMe
MeO
8'-epi-cleomiscosin A: R1=H, R2=CH
2
OH
(RA
a
, 12.8F; ref.85)
Cleomiscosin A: R1=CH
2
OH, R2=H
(RA
a
, 0.8F; ref.85)
O O H
Clu
O
O
Aloesin
(Competitive; RA
b
, 0.2F; ref.80)


Int. J. Mol. Sci. 2009, 10


2455
3.2. Benzaldehyde and Benzoate Derivatives
In the past decade, a large number of benzaldehyde and benzoate derivatives have been isolated
from plants and identified as tyrosinase inhibitors, including benzoic acid, benzaldehyde, anisic acid,
anisaldehyde, cinnamic acid, and methoxycinnamic acid from the roots of Pulsatilla cernua [86],
4-substituted benzaldehydes from cumin [87], 2-hydroxy-4-methoxybenzaldehyde from roots of
Mondia whitei [88], p-coumaric acid from the leaves of Panax ginseng [89], hydroxycinnamoyl
derivatives from green coffee beans [90], and vanillic acid and its derivatives from black rice bran [91].
The aldehyde group is known to react with biologically important nucleophilic groups such as
sulfhydryl, amino, and hydroxyl groups. The tyrosinase inhibitory mechanism of benzaldehyde-type
inhibitors comes from their ability to form a Schiff base with a primary amino group in the enzyme
[92-93]. In contrast, benzoate inhibits tyrosinase by a copper chelating mechanism and belongs to a
typical HA-type acid tyrosinase inhibitor, whose inhibitory mechanism involves the interaction
between the non-ionized form of the inhibitor and the copper in the active site of the enzyme [94]. In
terms of inhibitory strength, all the naturally occurring benzaldehyde and benzoate derivatives listed
above showed only weak-to-moderate tyrosinase inhibitory activity while none are stronger than kojic
acid. Recently, flourobenzaldehydes [95], methyl trans-cinnamate [96], salicylic acid [97],
hydroxybenzaldehydes [98], and 4--D-glucopyranosyloxybenzoate [99] were also proved to be weak
tyrosinase inhibitors with one to two orders of magnitude of lower antityrosinase activity than that of
kojic acid. The most potent natural inhibitor of benzaldehyde type was not identified in plants but in a
fungus. Protocatechualdehyde (Figure 4d) was isolated from the fruiting body of Phellinus linteus and
exhibited 7.8-fold more tyrosinase inhibitory activity than that of kojic acid [100]. It is worth noting
that its analog, protocatechuic aldehyde, with two methoxyl groups replacing the two hydroxyl groups
showed one order of magnetite of lower activity than that of protocatechualdehyde [101]. Moreover,
another analog, protocatechuic acid isolated from black rice bran with a benzoate skeleton, showed
another one order of magnetite of lower activity [91]. Thus, it is indicated that both the o-dihydroxyl
group and the aldehyde group of the protocatechualdehyde structure play important roles in exerting
its inhibition activity. On the other hand, to improve the inhibitory strength of the benzaldehyde-type
inhibitors, some derivatives were chemically synthesized and assayed to determine their inhibitory
activity. 4-Vinylbenzaldehyde [102], 4-alkylbenzaldehyde [103-104], 2-hydroxy-4-isopropyl-
benzaldehyde [105], 3,4-dihydroxybenzaldehyde-O-ethyloxime (Figure 4d) [106], and 4-butyl-
benzaldehyde thiosemicarbazone [107] were successfully synthesized and showed 35-, 100-, 350-,
1500-, and 2000-fold, respectively, more potent activity than that of the precursor benzaldehyde.
However, advanced data for cell cytotoxicity and cellular melanogenesis inhibition are lacking for
these improved benzaldehyde derivative inhibitors.
Gallic acid (3,4,5-trihydroxybenzoate) has been isolated and identified as a tyrosinase inhibitor
from many plants, and its inhibitory mechanism together with those of its ester derivatives has been
well studied by Kubo et al. [108-110]. They found that gallic acid inhibited diphenolase activity of
mushroom tyrosinase with a IC
50
value of 4500 M, which is 100-fold lower than that of kojic acid. In
addition, gallic acid itself and its short alkyl chain esters (<C10) were oxidized by mushroom
tyrosinase as substrates, but the long-chain alkyl chain esters (>C10) inhibited the enzyme without
being oxidized. Gallic acid was also found to be very toxic to melanoma cells with cytotoxicity
Int. J. Mol. Sci. 2009, 10


2456
comparable to that of hydroquinone [111]. Recently, Nithitanakool et al. isolated both gallic acid and
its methyl derivative from seed kernels of Mangifera indica, and determined that both compounds
were poor inhibitors against diphenolase activity of mushroom tyrosinase with 300- and 30-fold,
respectively, lower activity than that of kojic acid [112]. These results were consistent with previous
studies. In spite of the poor inhibitory activity of gallic acid itself, some compounds with gallate
moiety were found to inhibit mushroom tyrosinase more effectively. Some flavonoids with gallate
moiety bonded to the 3-hydroxyl position, including GCG [(+)-gallocatechin-3-O-gallate] and EGCG
[(-)-epigallocatechin-3-O-gallate], were isolated from green tea leaves and showed stronger inhibitory
activity [113]. Similarly, three tyrosyl gallates were synthesized and showed stronger inhibitory
activity [114]. In recent times, 1,2,3,4,6-pentagalloylglucopyranose isolated from the seed kernels of M.
indica [112] and the roots of Paeonia suffruticosa [115], respectively, was found to be 16-fold more
active than gallic acid and inhibited monophenolase activity of mushroom tyrosinase with a non-
competitive mode.
Figure 5. Chemical structures of selected tyrosinase inhibitors belonging to long-chain
lipids (a) or steroids (b). RA
a
and RA
b
have the same meanings as those of Figure 3.
(a) Long-chain lipids
(b) Steroids
2(2S)-Hydroxyl-7(E)-
tritriacontenoate
(RA
a
, 12.3F; ref.120)
Trilinolein
(Non-competitive; RA
a
, 1.7F; ref.116)
O H
O H
OH
OH
O H
O H
OH
COOH
O H
OH
OH
O
Stigmast-5-ene-3,26-diol
(RA
a
, 7.2F; ref.119)
3,21,22,23-Tetrahydroxy-
cycloart-24(31),25(26)-diene
(RA
a
, 12.6F; ref.121)
Arjunilic acid
(RA
a
, 16.7F; ref.123)
17-Ethylsteroid
(RA
a
, 9.8F; ref.129)
(CH
2
)
24
CH
3
O
O
OH
CH
2
O C
O
(CH
2
)
7
(CH
2
)
4
CH
3
CHO
C
O
(CH
2
)
7
(CH
2
)
4
CH
3
CH
2
O C
O
(CH
2
)
7
(CH
2
)
4
CH
3
(a) Long-chain lipids
(b) Steroids
2(2S)-Hydroxyl-7(E)-
tritriacontenoate
(RA
a
, 12.3F; ref.120)
Trilinolein
(Non-competitive; RA
a
, 1.7F; ref.116)
O H
O H
OH
OH
O H
O H
OH
COOH
O H
OH
OH
O
Stigmast-5-ene-3,26-diol
(RA
a
, 7.2F; ref.119)
3,21,22,23-Tetrahydroxy-
cycloart-24(31),25(26)-diene
(RA
a
, 12.6F; ref.121)
Arjunilic acid
(RA
a
, 16.7F; ref.123)
17-Ethylsteroid
(RA
a
, 9.8F; ref.129)
(CH
2
)
24
CH
3
O
O
OH
CH
2
O C
O
(CH
2
)
7
(CH
2
)
4
CH
3
CHO
C
O
(CH
2
)
7
(CH
2
)
4
CH
3
CH
2
O C
O
(CH
2
)
7
(CH
2
)
4
CH
3


3.3. Long-chain Lipids and Steroids
Recently, several lipids were purified from natural sources and exhibited tyrosinase inhibitory
activity. A triacylglycerol, trilinolein (Figure 5a), was isolated from sake lees, which are byproducts of
sake production, and proved to be as potent as kojic acid for inhibition of diphenolase activity of
mushroom tyrosinase [116]. Based on the findings, including inability of copper chelating, lack of free
radical scavenging, and kinetically non-competitive inhibition of the inhibitor, the inhibitory
mechanism was proposed by binding of the compound to some site of the tyrosinase, except the
catalytic site. A glycosphingolipid, soyacerebroside I, which is composed of a sphingoid base skeleton,
an amide aliphatic long-chain fatty acid, and a -glucopyranose moiety, was isolated from the leaves
of Guioa villosa and found to inhibit monophenolase and diphenolase activity of mushroom tyrosinase
Int. J. Mol. Sci. 2009, 10


2457
with half-activity of kojic acid [117] while another glycosphinogolipid, cerebroside B, from Phellinus
linteus showed no inhibitory activity against the enzyme [100]. In addition, trans geranic acid from
Cymbopogon citrates (lemongrass) was recently identified as a tyrosinase inhibitor but with only one-
tenth of the inhibitory activity of kojic acid [118].
In addition to long-chain lipids, some steroids were also determined to be tyrosinase inhibitors.
Many studies in this field were contributed by the research group of Choudhary and Khan. Three
steroids isolated by these authors from the aerial parts of Trifolium balansae showed higher
diphenolase inhibitory activity toward mushroom tyrosinase than that of kojic acid [119]. Among the
steroids, stigmast-5-ene-3,26-diol (Figure 5b) was 7-fold more active. The authors also found that a
long-chain ester, 2(2S)-hydroxyl-7(E)-tritriacontenoate (Figure 5a), from Amberboa ramose
exhibited 12.3-fold more inhibition against the diphenolase activity of mushroom tyrosinase compared
to the standard kojic acid [120]. On the other hand, the derivative containing a D-galactopyranosyl
moiety at C2 was found to lose one order of magnitude on its tyrosinase inhibitory activity. Like most
cases of tyrosinase inhibitors, the presence of the bulky and hydrophilic D-galactopyranosyl moiety
interferes with the entrance of the molecule into the active site of the enzyme, thus reducing its
inhibitory activity. From the same plant, eight cycloartane triterpenoids were isolated at the same time.
A triterpenoid, 3,21,22,23-tetrahydroxycycloart-24(31),25(26)-diene (Figure 5b), exhibited an
extremely potent inhibition (12.6-fold) against diphenolase activity of mushroom tyrosinase when
compared with kojic acid [121]. The same research group also purified some triterpenoid glycosides
from the roots of Astragalus taschkendicus. However, the triterpenoid glycosides showed only
inhibitory activity equal to that of kojic acid against mushroom tyrosinase [122]. In advance,
Choudhary and Khan purified nine pentacyclic triterpenes from the aerial part of the plant
Rhododendron collettianum, and all the triterpenes showed potent diphenolase inhibitory activity of
mushroom tyrosinase. Among them, arjunilic acid (Figure 5b) was the strongest tyrosinase inhibitor,
which was 16.7-fold inhibitory activity of kojic acid [123]. In addition to triterpenoids, the authors also
purified many diterpenoids from the aerial parts of Aconitum leave [124]. However, most diterpenoids
did not inhibit tyrosinase activity. Only one compound, lappaconitine hydrobromide, revealed activity
similar to that of kojic acid [125]. Similar to the diterpenoids, few monoterpenoids were found to be
strong tyrosinase inhibitors; only a monoterpenoid, crocusatin-K, isolated from the petals of Crocus
sativus, was proved to display inhibitory activity equal to that of kojic acid [126]. More recently, Wu
et al. isolated four new sesquiterpenes from the leaves and stems of Chloranthus henryi [127] and two
new sesquiterpenes dimmers from the leaves of Chloranthus tianmushanensis [128], and found that
these sesquiterpenes showed either no or weak tyrosinase inhibitory activity. In addition to the original
steroids from plants, two 11-hydroxylated steroid metabolites were isolated from the fungus
Cunninghamella elegans cultivations feeding with 17-ethynyl- or 17-ethylsteroids (Figure 5b) and
showed 2.9- and 9.8-fold higher tyrosinase inhibitory activity, respectively, than that of kojic acid
[129]. Regarding the difficult permeability of skin, these hydrophobic steroid or long-chain lipid
inhibitors seems to have superior potential in the development as skin-whitening agents due to their
easier cellular permeability. However, all these lipid inhibitors are lack of cellular or clinical assays for
determining their depigmentation activity and remain less utilized by the cosmetics industry until now.
Int. J. Mol. Sci. 2009, 10


2458
3.4. Other Natural and Synthetic Inhibitors
Other inhibitors from natural sources. Anthraquinones from different plant sources have been widely
used since ancient times due to their laxative and cathartic properties. In addition, this class of
compounds has shown a wide variety of pharmacological activities, such as antiinflammatory, wound
healing, analgesic, antipyretic, antimicrobial, and antitumor activities. Recently, an anthraquinone,
physcion (1,8-dihydroxy-2-methoxy-3-methylanthraquinone, Figure 6a), was found to show similar
tyrosinase inhibitory activity with that of kojic acid [130]. Interestingly, another anthraquinone, 1,5-
dihydroxy-7-methoxy-3-methylanthraquinone (Figure 6a), with hydroxyl and methoxyl groups at
different positions compared with those of physcion exhibited a 72-fold increase on antityrosinase
activity [131]. Therefore, it is worthy to systematically evaluate the structure-activity relationship
between the functional groups attached to the anthraquinone skeleton and the antityrosinase activity. In
addition, many lignans isolated from the roots of Vitex negundo showed higher tyrosinase inhibitory
activity than kojic acid, while the most active lignan from the plant was (+)-lyoniresinol (Figure 6a),
whose activity was 5.2-fold higher than that of kojic acid [132]. In addition to the inhibitors from
plants, marine beings inhabit virtually any environment in the sea, and they have been shown to
produce novel substances with utilities in fine chemicals, drug, and cosmetics products, including
tyrosinase inhibitors. One phloroglucinol derivative, dieckol, was isolated from a marine brown alga,
Ecklonia stolonifera, and displayed three times more activity than that of kojic acid [133]. In addition,
a marine-derived fungus Myrothecium sp. was found to contain 6-n-pentyl--pyrone (Figure 6a),
which was a potent tyrosinase inhibitor with 9.6-fold inhibitory activity of kojic acid [134]. Recently,
another tyrosinase inhibitor was purified from a marine bacteria, Trichoderma viride strain H1-7, and
showed competitive inhibition toward monophenolase activity of mushroom tyrosinase through
binding to a copper active site of the enzyme [135].
Figure 6. Chemical structures of other tyrosinase inhibitors from natural (a) or synthetic (b)
sources. RA
a
and RA
b
have the same meanings as those of Figure 3.
(a) Other natural tyrosinase inhibitors (b) Other synthetic tyrosinase inhibitors
Physcion: R1=OH, R2=R4=H, R3=OCH
3
(RA
a
, 1.0F; ref.130)
Physcion Analog: R1=R3=H, R2=OCH
3
, R4=OH
(RA
a
, 72.4F; ref.131)
R2
R4
O
O
OH R1
R3
O H
OH
CH
2
OH
CH
2
OH
MeO
OMe
MeO OMe
(+)-Lyoniresinol
(RA
a
, 5.2F; ref.132)
O O
Oxadiazole derivative 3e
(RA
a
, 7.6F; ref.145)
N NHR
R1
PTU: R=H, R1=H
(RA
a
, 41.7F; ref.137)
PTU Analog: R=OH, R1=S
(RA
a
, 259F; ref.137)
O
N N
N
Br
N
O
O
Oxazolone derivative 7
(RA
a
, 13.6F; ref.146)
O O
O O
NH
2
Tetraketone derivative 11
(RA
a
, 8.3F; ref.147)
O H OH
4,.4-Dihydroxybiphenyl
(Competitive; RA
a
,
12.6F; ref.151)
N
H
S
O
S-Phenetyl N-
phenylthiocarbamate
(RA
a
, 43.6F; ref.154)
6-n-Pentyl--pyrone
(RA
a
, 9.6F; ref.134)
(a) Other natural tyrosinase inhibitors (b) Other synthetic tyrosinase inhibitors
Physcion: R1=OH, R2=R4=H, R3=OCH
3
(RA
a
, 1.0F; ref.130)
Physcion Analog: R1=R3=H, R2=OCH
3
, R4=OH
(RA
a
, 72.4F; ref.131)
R2
R4
O
O
OH R1
R3
O H
OH
CH
2
OH
CH
2
OH
MeO
OMe
MeO OMe
(+)-Lyoniresinol
(RA
a
, 5.2F; ref.132)
O O
Oxadiazole derivative 3e
(RA
a
, 7.6F; ref.145)
N NHR
R1
PTU: R=H, R1=H
(RA
a
, 41.7F; ref.137)
PTU Analog: R=OH, R1=S
(RA
a
, 259F; ref.137)
O
N N
N
Br
N
O
O
Oxazolone derivative 7
(RA
a
, 13.6F; ref.146)
O O
O O
NH
2
Tetraketone derivative 11
(RA
a
, 8.3F; ref.147)
O H OH
4,.4-Dihydroxybiphenyl
(Competitive; RA
a
,
12.6F; ref.151)
N
H
S
O
S-Phenetyl N-
phenylthiocarbamate
(RA
a
, 43.6F; ref.154)
6-n-Pentyl--pyrone
(RA
a
, 9.6F; ref.134)


Other inhibitors from synthetic sources. For smaller molecules, it is easier to rationally design the
structures of the molecules by a chemically synthetic method. Improving the tyrosinase inhibitory
Int. J. Mol. Sci. 2009, 10


2459
activity of a known inhibitor by properly changing the substitute groups on its structure is expected. N-
Phenylthiourea (PTU, Figure 6b) is a well-known inhibitor for inhibiting diphenolase enzyme that
belongs to the type-3 copper protein group. The sulfur atom of the compound binds to both copper ions
in the active site of the enzyme and blocks enzyme activity [136]. Criton and Le Mellay-Hamon
synthesized a series of analogs of N-phenylthiourea and put into assays of the inhibitory activity of the
analogs against diphenolase activity of mushroom tyrosinase. They found that when the amino group
and the sulfur moieties of N-phenylthiourea were replaced by N-hydroxylamine and oxygen,
respectively, the resulting compound (Figure 6b) exhibited six times more activity than that of
N-phenylthiourea [137]. In another study, the same authors synthesized N-(phenylalkyl)cinnamides
derived from the coupling cinnamic acid with phenylalkylamines and found that two synthetic
compounds showed higher inhibitory activity than that of kojic acid [138]. Similarly, Kang et al.
designed a series of compounds by combining the structures of two putative tyrosinase inhibitors, kojic
acid and caffeic acid, to form new inhibitors, which showed antidiphenolase activity equal to that of
kojic acid toward mushroom tyrosinase but enhanced depigmentation activity in melanoma cells [139].
In addition, Shiino et al. synthesized analogs of cupferron, which is a well-known metal chelating
agent and inhibits competitively both monophenolase and diphenolase activity of mushroom tyrosinase
[140], and found that N-substituted-N-nitrosohydroxylamines inhibited mushroom tyrosinase by
interacting with the copper ions at the active site of the enzyme [141]. As removal of nitroso or
hydroxyl moiety completely diminished the inhibitory activity, both functional groups were suggested
to be essential for chelating activity. Among the derivatives, the authors found that N-hydroxybenzyl-
N-nitrosohydroxylamines showed the best inhibitory activity, which is comparable with that of kojic
acid [142]. In advance, they recently reported that these N-substituted-N-nitrosohydroxylamines
inhibited mushroom tyrosinase in a pH-dependent manner due to the non-ionized, electrically neutral
form of the compounds [143]. Another research group led by Khan also did a lot of successful work
for developing new tyrosinase inhibitors by chemically synthetic methods. The discovered new types
of inhibitors included sildenafil [144], oxadiazole [145], oxazolones [146], and tetraketones types [147]
(Figure 6b). The strongest inhibitor of each type list above was between 7- and 14-fold stronger than
kojic acid. On the other hand, Kim et al. focused on developing new type inhibitors containing a
selenium atom, which is an essential biological trace element and an integral component of several
enzymes. The use of selenium as a nutritional supplement has been popularized recently due to its
potential roles as an antioxidant and an anticancer agent. The authors demonstrated that some 1,3-
selenazol-4-one derivatives [148], selenourea derivatives [149], and selenium-containing
carbohydrates [150] possessed inhibitory activity similar to that of kojic acid against diphenolase
activity of mushroom tyrosinase. However, these selenium-containing derivatives were applied in
melanogenesis inhibition assays in melanoma cells; most compounds showed both cytotoxicity and
depigmenting effects and had restrictions in applications to cosmetics or foods.

Some simple phenyl and biphenyl compounds were also synthesized and identified as potent
tyrosinase inhibitors. 4,4'-Dihyldroxybiphenyl (Figure 6b) showed 12-fold more monophenolase
inhibitory activity of mushroom tyrosinase than that of kojic acid with a competitive inhibition mode
[151] while its glucoside derivatives from the fruit of Pyracantha fortuneana displayed only low
inhibitory activity [152]. In addition to directly inhibiting tyrosinase activity, 4,4'-dihyldroxybiphenyl
Int. J. Mol. Sci. 2009, 10


2460
was also found to suppress several cellular key parameters in the melanogenic pathway by
downregulating the cAMP-dependent protein kinase K signaling pathway and decreasing gene
expression of microphthalmia transcription factor, which in turn suppressed tyrosinase expression
[153]. In addition, S-phenyl N-phenylthiocarbamate (Figure 6b) [154] and 4-(2',4'-dihydroxyphenyl)-
(E)-3-buten-2-one [155] were recently found to be 44-fold and 6-fold, respectively, more active than
kojic acid toward diphenolase activity of mushroom tyrosinase. However, although huge numbers of
synthetic inhibitors were successful in inhibiting tyrosinase activity, few have been confirmed in
melanogenesis inhibiting activity in cells or skin models.
3.5. Irreversible Inactivators
In contrast to the huge number of reversible inhibitors has been identified, rarely irreversible
inhibitors of tyrosinase were found until now. These irreversible inhibitors, which are also called
specific inactivators, can form irreversibly covalent bond with the target enzyme and then inactivate it.
However, the tyrosinase irreversible inhibitors are different from non-specifically irreversible enzyme
inactivators such as acids or bases, which destroy all protein structures. Instead, they are generally
specific for tyrosinase and do not inactivate all proteins; they work by specifically altering the active
site of the enzyme. The substrate reaction kinetics method described by Tsou has been widely used in
studies of inactivation of various enzymes by different types of inactivation or inhibitions [156]. As
shown in Scheme 2, irreversible inhibitors form a reversible non-covalent complex with the enzyme
(EI or ESI), and this then reacts to produce the covalently modified dead-end complex E
i
. The rate at
which E
i
is formed is called the inactivation rate or k
inact
. It is worth noting that irreversible inhibitors
display time-dependent inhibition, and their potency therefore cannot be characterized by an IC
50
value.
This is because the amount of active enzyme in a given concentration of irreversible inhibitor will be
different depending on how long the inhibitor is pre-incubated with the enzyme.
Scheme 2. Reaction mechanism of irreversible inhibitors. E and E
i
are the enzyme and the
inactivated enzyme, respectively; S, I, and P are the substrate, inhibitor, and product,
respectively; ES, EI and ESI are the intermediates.

E + S ES
E + P
k
inact

E
i

+
I
EI
+
I
ESI
K
1

K
I

K
I
'
Int. J. Mol. Sci. 2009, 10


2461
Captopril (Figure 7), an antihypertensive drug [(2S)-1-(3-mercapto-2-methylpropionyl)-L-proline],
forms both a copper-captopril complex and a disulfide bond between captopril and cysteine-rich
domains at the active site of tyrosinase [157]. Accordingly, the drug is able to prevent melanin
formation by irreversibly inhibiting monophenolase and diphenolase activity of mushroom tyrosinase
in a non-competitive and competitive manner, respectively, as well as by scavenging the generated o-
quinones to form a colorless conjugate. The kinetic study showed that the value of the rate inactivation
constant k
inact
was 1.98 x 10
-4
M
-1
s
-1
. H
2
O
2
is also known as an inactivator of several copper-
containing enzymes, such as dopamine -monooxygenase [158] and mushroom tyrosinase [159]. It
was proposed that high concentrations of H
2
O
2
could oxidize a methionine residue in position 374 of
the mushroom tyrosinase active site to methionine sulfoxide, and this event would inactivate the
enzyme [160]. Chen et al. discovered two other irreversible inhibitors of tyrosinase, including
cetylpyridinium chloride [161] and 3,5-dihydroxyphenyl decanoate [162] (Figure 7). The fluorescence
spectroscopic results demonstrated that cetylpyridinium chloride can bind to the tyrosinase molecule
and induce enzyme conformational changes, which undergo a slow irreversible inactivation of the
enzyme. The kinetic study determined the values of the inactivation constants of the inhibitor toward
the free enzyme and enzyme-substrate complex to be 3.957 and 6.078 x 10
-3
s
-1
, respectively. On the
other hand, the inhibition mechanism of 3,5-dihydroxyphenyl decanoate was irreversible and
competitive/uncompetitive mixed type inhibition with varied inactivation constants ranging from 1.93
to 7.912 x 10
-3
s
-1
. Recently, p-hydroxybenzyl alcohol showed binding capability of mushroom
tyrosinase and irreversibly inhibited the enzyme [163]. It was demonstrated that p-hydroxybenzyl
alcohol (Figure 7) exhibited an inhibitory effect on melanogenesis in melanoma cells at non-cytotoxic
concentrations. The suppression of melanin synthesis by the compound was attributed to its behavior
in directly inhibiting cellular tyrosinase activity without effects on the expression level of tyrosinase
mRNA. In another study, it is very interesting to find that hen egg white lysozyme (HEWL) inhibited
mushroom tyrosinase with a reversibly and irreversibly mixed inhibition mechanism [164]. The
authors suggested that HEWL binds to glycoside linkage in tyrosinase and induces its conformational
change. Some of the bound HEWL continues to non-specifically cleavage glycosidic linkages and
induces irreversible inhibition while the other portion of the HEWL-tyrosinase complex restored the
diphenolase activity of tyrosinase after depletion of HEWL.
Figure 7. Chemical structures of irreversible tyrosinase inhibitors.
Captopril
Cetylpyridiniumchloride
3,5-Dihydroxyphenyl decanoate
p-Hydroxybenzyl alcohol
N
O S H
COOH
N
+
Cl
O H O
OH
O
OH
OH
Captopril
Cetylpyridiniumchloride
3,5-Dihydroxyphenyl decanoate
p-Hydroxybenzyl alcohol
N
O S H
COOH
N
+
Cl
O H O
OH
O
OH
OH

Int. J. Mol. Sci. 2009, 10


2462
Among the irreversible inhibitors, suicide substrates belong to a special class. It is known that
tyrosinase could be irreversibly inhibited by its o-diphenol substrates, such as L-dopa and catechol
[165]. These substrates were also named suicide substrates or mechanism-based inhibitors. The
mechanism of the suicide substrate has been extensively studied by Waley [166], who proposed a
simple branched reaction pathway as Scheme 3, in which an intermediate Y may give either an active
enzyme and product, or an inactive enzyme. In the scheme, the intermediate Y has a choice of reaction,
governed by the partition ratio r, where r = k
+3
/k
+4
. The r value is called the molar proportion for
inactivation, i.e., the number of molecules of inhibitors required to completely inactivate one molecule
of the enzyme, and may be determined by plotting the fractional activity remaining against the ratio of
the initial concentration of inhibitor to that of enzyme. The intercept on the abscissa is 1 + r in the plot,
when r > 1 [167]. As in general irreversible inhibitors, the inhibitory strength of a suicide substrate is
also not determined by an IC
50
value but expressed by its r value, where a smaller r value of a suicide
substrate means fewer inhibitor molecules are needed to inactivate all enzyme activity and being more
powerful inhibition.
Scheme 3. Action mechanism for suicide substrate. E and E
i
are the enzyme and
inactivated enzyme, respectively; P is the product; X is the first intermediate, and Y is
another intermediate.

In addition to the kinetic reaction mechanism, Land et al. recently proposed a model to explain the
detailed molecular reaction mechanism of the suicide inactivation of tyrosinase based on the chemical
structures of substrates and crystallographic tyrosinase [168]. In their model, the inactivation of
tyrosinase during catechol oxidation is due to the catechol being capable of an alternative cresolase
(monophenolase) presentation (Figure 8). Oxygen addition to the catechol ring by cresolase activity
generates an intermediate product that can undergo deprotonation and reductive elimination. This
deprotonation leads to the inactivation of the enzyme by formation of a copper atom, which does not
bind to the histidine ligands. According to the model, suicide inactivation of tyrosinase depends on
anomalous catechol oxidation and the consequence of the formation of zero-valency copper. Their
model can explain well the experimental observation that half of the copper is lost from the active site
during catechol inactivation. Moreover, the authors in advance used hplc/mass to identify the
hydroquinone product in the suicide inactivation of mushroom tyrosinase with 4-methylcatechol as a
substrate [169]. Very interestingly, tyrosinase has the following two unique properties: (1) Tyrosinase
is able to catalyze oxidations of both monophenols (cresolase activity) and diphenols (catecholase
activity) to o-quinones, and (2) tyrosinase exhibits suicide inactivation during the oxidation of
E + I X Y E + P
k
+1

k
-1

k
+2

k
+3

k
+4

E
i

Int. J. Mol. Sci. 2009, 10


2463
catechols. In the Land cresolase-dependent model, the two properties are neatly connected, and suicide
inactivation is the result of catecholic substrates being processed by the cresolase route.
Figure 8. Molecular reaction mechanism of suicide inactivation of tyrosinase by the
oxidation of an o-diphenol substrate. The curly arrows shows the effect of deprotonation
leading to the reduction of copper from bivalent to zero-valent form, elimination of an o-
quinone and inactivated tyrosinase [168].
OH O
O
R
Cu
2+
N
N
N
O
O
Cu
2+ N
N
N
Cu
2+
N
N
N
OH-
Cu
2+ N
N
N
O
O
R
OH-
N
N
Cu
2+ N
N
N
O
O
R
O
N
H
+
Cu
H
2
O
N
N
Cu
2+
N
N
N
N
H
2
O
Cu
2+
N
N
N
Cu
2+ N
N
N
OH-
oxy-tyrosinase
R
OH
O H
R
OH
OH
met-tyrosinase
Cu
2+
N
N
N
Cu
2+ N
N
N
O
O
R
O H
OH-
H
+
inactivated tyrosinase
+ Cu
Catecholase catalysis of
catechols
Cresolase catalysis of
catechols
Cu
2+
N
N
Cu
2+ N
N
N
O
O
R
O
OH-
H
+
H
N
R
O
O
OH O
O
R
Cu
2+
N
N
N
O
O
Cu
2+ N
N
N
Cu
2+
N
N
N
OH-
Cu
2+ N
N
N
O
O
R
OH-
N
N
Cu
2+ N
N
N
O
O
R
O
N
H
+
Cu
H
2
O
N
N
Cu
2+
N
N
N
N
H
2
O
Cu
2+
N
N
N
Cu
2+ N
N
N
OH-
oxy-tyrosinase
R
OH
O H
R
OH
OH
met-tyrosinase
Cu
2+
N
N
N
Cu
2+ N
N
N
O
O
R
O H
OH-
H
+
inactivated tyrosinase
+ Cu
Catecholase catalysis of
catechols
Cresolase catalysis of
catechols
Cu
2+
N
N
Cu
2+ N
N
N
O
O
R
O
OH-
H
+
H
N
R
O
O


Although both the kinetic and the molecular reaction mechanism of suicide inactivation of
tyrosinase have been well studied, potent suicide substrates have rarely been discovered. Recently, we
found that two isoflavones, 7,8,4'-trihydroxyisoflavone and 5,7,8,4'-tetrahydroxyisoflavone (Figure 3h),
were potent and unique suicide substrates of mushroom tyrosinase with low partition ratios, low
Int. J. Mol. Sci. 2009, 10


2464
Michaelis constants, and high maximal inactivation rate constants [54]. The partition ratios between
molecules of suicide substrate in the formation of the product and in the inactivation of the enzyme
were determined to be 81.7 and 35.5, for 7,8,4'-trihydroxyisoflavone and 5,7,8,4'-tetrahydroxy-
isoflavone, respectively. In the reviewed literature, 5,7,8,4'-tetrahydroxyisoflavone is the most potent
suicide substrate of mushroom tyrosinase until now and has high potential in application as a skin-
whitening agent. This compound was in advance creamed in the proper condition [170] and showed
higher depigmenting activity than that of ascorbic acid 2-glucoside (AA2G) in an in vivo assay
conducted with 60 volunteers by our laboratory (unpublished data).
4. Conclusions
The commercial availability of mushroom tyrosinase plays a critical role in tyrosinase inhibitor
studies, and most research has been conducted with this enzyme, which is well studied and easily
purified from the mushroom A. bisporus. No matter in terms of inhibitory strength, inhibitory
mechanism, chemical structures, or the sources of the inhibitors, the search for new inhibitors based on
mushroom tyrosinase has been so successful that various different types of inhibitors have been found
in the past 20 years. This success is also based on several fundamental studies in different fields, such
as the early finding and the detailed study of tyrosinase as key in melanin biosynthesis, and the deep
research into the biochemical, kinetic, reaction mechanistic, and structural aspects of the enzyme.
In spite of this success, it is obvious that much work in this field is still needed. First of all, the
commonly used mushroom tyrosinase has some limitations related to the applications of the mushroom
tyrosinase inhibitors for human use due to the differences between the fungal tyrosinase and the human
one. The mushroom tyrosinase is a cytosol enzyme while the human tyrosinase is membrane bonded
[9]. In addition, mushroom tyrosinase is a tetramer in contrast to the monomer type of the human
enzyme, which is highly glycosylated during its complex maturation process [21]. However,
information regarding the discovered inhibitors to human tyrosinase is very limited; thus, the
biochemical information related to kinetic and mechanistic characterizations and to the structure
activity relationship between the inhibitors and the human tyrosinase needs further elucidation. To date,
no tyrosinase inhibitory study has been performed with human tyrosinase due to the lack of a purified
enzyme. Although some studies used a crude extract of human melanocytes as the enzyme source, it is
not possible to study the detailed kinetic and mechanistic characterizations of the inhibitors to the
specific enzyme in the very complex system. As the primary genetic information of human tyrosinase
has been known, utilizing modern genetic engineering to produce recombinant human tyrosinase with
suitable properties for easier purification could be possible. Using the purified human tyrosinase to
elucidate inhibitory study will aid us considerably in developing the next generation of tyrosinase
inhibitors. Moreover, the easier availability of human tyrosinase is also important for the resolution of
the X-ray crystallographic structure of the enzyme. Nearly all molecular docking experiments of
tyrosinase inhibitors were performed with the recently resolved crystallographic structure of tyrosinase
from Streptomyces glaucescens [20]; however, the membrane-bonded property of human tyrosinase is
expected to show something different in the enzyme structure, which is the prime criterion for inhibitor
binding. Thus, the X-ray crystallographic structure of human tyrosinase is also needed and can shed
Int. J. Mol. Sci. 2009, 10


2465
more light on the action mechanism of human tyrosinase and will be helpful in designing more suitable
tyrosinase inhibitors for human use.
On the other hand, the use of the inhibitors is primary in the cosmetic industry due to their skin-
whitening effects. Since a huge number of tyrosinase inhibitors have been developed, clarifying the
validation of these inhibitors in skin-whitening efficiency has become more urgent. Most inhibitors
have rarely been incorporated in topically applied cosmetics or cosmeceuticals, often due to a lack of
parallel human clinical trials. In addition, most tyrosinase inhibitors listed above are not currently
commercially available, especially those from natural sources, and this limits their further evaluation
in an in vivo study, where usually a large amount is needed for a tested inhibitor. In conclusion, more
concrete studies of the found inhibitors with a human clinical point of view are required, and in our
experience, this often needs the help and cooperation of cosmetic or biotechnology companies.
Acknowledgements
The author wants to thank Dr. W.T. Wu, who is the head of the College of Engineering, National
Cheng Kung University, for his long-term, continuous help in advising and encouraging our research.
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